Nitric oxide (NO) is a biologically active compound derived from L-arginine. NO has disparate physiological roles, from intercellular signaling to toxicity, depending upon its concentration and location within an animal (reviewed in Moncada et al., Pharm. Rev., 43(2) 109-42 (1991); Morris & Billiar, Am. Phsiol. Soc., E829-37 (1994); Schmidt & Walter, Cell, 78, 919-25 (1994); Fedlman et al., C. & E. N., 71, 26-38 (1993)).
Nitric oxide is normally produced by the vascular endothelium, but because of a very short half-life (t1/2 in seconds), it diffuses only to the adjacent smooth muscle where it causes relaxation of vascular smooth muscles via the activation of soluble guanylate cyclase (Moncada et al., Pharmacol. Rev., 43, 109-42 (1991)). Nitric oxide released toward the lumen assists in preventing platelet adherence. The small amounts of nitric oxide derived from endothelial cells is produced in an ongoing fashion (Palmer et al., Nature, 327, 524-26 (1987); Ignarro et al., Proc. Natl. Acad. Sci. USA, 84, 9265-69 (1987)) by an enzyme (eNOS), which is located primarily on microsomal and plasma membranes.
Nitric oxide is known to be important to vascular integrity and the prevention of atherosclerotic lesions by promoting vasodilation (Palmer et al., supra; Ignarro et al., supra), inhibiting platelet adherence and aggregation (Radomski et al., Br. J. Pharmacol., 92, 639-46 (1987)), inhibiting vascular smooth muscle (Nunokawa et al., Biochem. Biophys. Res. Com., 188, 409-15 (1992)) and fibroblast (Werner-Felmayer et al., J. Exp. Med., 172, 1599-1607 (1990)) cellular proliferation.
Several conditions can result in decreased NO production within an individual. For example, in certain forms of diabetes, patients experience decreased production of NO. Furthermore, certain modes of steroid therapy inhibit production of NO. While decreased NO production is itself a cause for concern, such NO-deficiency contributes to unwelcome complications, for example, in healing wounds.
In some instances excessive production of nitric oxide is detrimental. For example, inducement of nitric oxide synthesis in blood vessels by bacterial endotoxins, such as, for example, bacterial lipopolysaccharide (LPS), and cytokines that are elevated in sepsis results in excessive dilation of blood vessels and sustained hypotension commonly encountered with septic shock (Kilbourn et al., Proc. Natl. Acad. Sci. USA, 87, 3629-32 (1990)). Overproduction of nitric oxide in lungs stimulated by immune complexes directly damages the lung (Mulligan et al., J. Immunol., 148, 3086-92 (1992)). Overproduction of nitric oxide in pancreatic islets impairs insulin secretion and contributes to the onset of juvenile diabetes (Corbett et al., J. Biol. Chem., 266, 21351-54 (1991)). Production of nitric oxide in joints in immune-mediated arthritis contributes to joint destruction (McCartney et al., J. Exp. Med., 178, 749-54 (1993)).
NO is synthesized from L-arginine through a reaction catalyzed by an enzyme referred to as nitric oxide synthase (NOS), of which there are three known isoforms. Two isoforms are constitutive NOS exhibiting strict dependence upon intracellular calcium and produce NO constitutively but in small quantities. One of these isoforms (nNOS or NOS-1) is localized primarily in the CNS, and the other (eNOS or NOS-3) is a membrane-bound protein found primarily in endothelial cells (Morris & Billiar, supra). The third NOS is inducible nitric oxide synthase (iNOS), which exhibits tonic catalytic activity for the life of the enzyme, and functions without requiring an increace in intracellular calcium concentration.
NOS-mediated catalytic production of NO from arginine requires the presence of a cofactor, tetrahydrobiopterin (BH.sub.4) (Tzeng et al., Proc. Nat. Acad. Sci. USA, 92, 11771-75 (1995); Tzeng et al., Surgery, 120(2), 315-21 (1995)). BH.sub.4 is necessary, in part, for maintaining the active structural configuration of the enzyme. Most cells express GTP cyclohydrolase I (GTPCH), which is the rate-limiting enzyme required for de novo BH.sub.4 synthesis. However, some tissues, such as vascular smooth muscle, express GTPCH only upon induction by cytokines (Tzeng et al., Surgery, 120(2), 315-21 (1995)).
iNOS expression increases dramatically in wound tissue (Carter et al., Biochem J., 304, 201-04 (1994); see also Shabani et al., Wound Healing Repair and Regeneration, 4(3), 353-62 (1996)). Furthermore, iNOS is expressed in various tissues in response to inflammatory stimulation by cytokines, and iNOS has been cloned and isolated from hepatocytes so stimulated. U.S. Pat. No. 5,468,630, issued to Billiar et al. on Nov. 21, 1995, discloses the human hepatocyte iNOS cDNA sequence. The plasmid pHINOS comprises the human iNOS coding region and was deposited under the terms of the Budapest Treaty on Nov. 20, 1992 and has the ATCC accession number 75358 (pHINOS) and ATCC accession number 69126 (pHINOS transformed in e. coli SOLR).
Sustained production of nitric oxide by iNOS has antimicrobial and antitumor functions. (see Granger et al., J. Clin. Invest., 81, 1129-36 (1989), and Hibbs et al., Science, 235, 473-76 (1987), respectively). Furthermore, when vascular smooth muscle cells are stimulated to express a iNOS enzyme by inflammatory cytokines, the large amounts of nitric oxide released contribute to the vasodilation and hypotension seen in sepsis (Busse and Mulsch, FEBS Letters, 265, 133-36 (1990)).
While termed "Nitric Oxide Synthase," iNOS-mediated catalysis produces other biologically active products. For example, N-hydroxyarginine, an intermediate byproduct of the iNOS enzyme (Stuehr et al., J. Biol. Chem., 266, 6259-63 (1991)), is known to induce cytostasis in proliferating cells in a dose dependent manner (Chenais et al., Biochem. Biophys. Res. Commun., 196, 1558-63 (1993)). Furthermore, NO itself acts in a dose-dependent manner, low concentrations being sufficient to mediate vasodilation while greater concentrations are required for cytostasis.
In light of its constitutive activity, the complex admixture of its biologically active products, and the capacity of its products to promote cytostasis among proliferating cells, delivery of exogenous iNOS appears an attractive method for treating disorders associated with hyperplasia. In fact, iNOS expression cassette transfer in vitro and in vivo has been demonstrated to achieve prophylactic and therapeutic relief from disorders associated with vascular occlusions (Tzeng et al., Mol. Med., 2(2), 211-25 (1996); see also International Patent Application No. WO 96/00006, (Billiar et al.)).
Following transplantation of graft tissue, a patient is at risk for rejection of the graft. Generally, graft rejection is characterized as either acute or chronic, based upon the mechanisms for rejection. As advances have been made in surgical technique, organ handling, management of acute graft rejection episodes, and control of post-operative infection, the threat of acute rejection of grafts has steadily declined (Paul & Tilney, "Alloantigen-Dependent Events in Chronic Rejection," in Transplantation Biology, Cellular and Molecular Aspects, Tilney et al., eds., Raven Pubs., Philadelphia, 567 (1996)). However, the threat of chronic graft rejection has not changed significantly, and it remains a significant risk to graft transplant procedures. For example, most kidney transplant failure not attributed to patient death is due to chronic rejection; roughly 60% of all heart transplant recipients and 50% of lung transplant recipients, respectively, develop manifestations of chronic rejection (Id.; see also Libbey, "Transplantation-Associated Arteriosclerosis, Potential Mechanisms," in Transplantation Biology, Cellular and Molecular Aspects, Tilney et al., eds., Raven Publishers, Philadelphia, 577 (1996); Hosenpud, Transplant Immunol., 1, 237-49 (1993)).
While the mechanisms causing the manifestation of chronic rejection remain poorly understood (Hosenpud, supra, page 237), the pathologic characteristics have been well defined. Vascular smooth muscle cells transform from a quiescent contractile phenotype to a rapidly proliferating phenotype. The proliferating smooth muscle cells invade the vascular lumen where they produce extracellular matrix material. Chronic rejection is thus characterized by progressive neointimal hyperplasia in vascular tissues, resulting in intimal thickening and eventual occlusion of vascular lumens in graft tissue (Hosenpud, supra; Ventura et al., Curriculum in Cardiology, 129 (4), 791-99 (1995); Libbey, supra). This presentation appears similar for transplanted heart, lung, and kidney tissue (Paul and Tilney, supra).
In comparison with naturally occurring arteriosclerosis (which is also produced by proliferating vascular smooth muscle cells, and is characterized by focal, and often eccentric, stenoses in large vessels) chronic rejection usually involves concentric arteriosclerosis extending over large regions of both large and small penetrating vessels. Moreover, chronic rejection arteriosclerosis develops extremely rapidly (Ventura et al., supra).
Efforts at preventing and treating allograft vasculopathy have met with only limited success. Advances in immunosuppression have failed to reduce the onset of chronic rejection (Ventura et al., supra, page 796). Some limited success has been reported with calcium-blocking agents and surgical intervention (Id., pages 796-97), but there is little evidence that these procedures influence long-term outcome (Hosenpud, supra). To date, re-transplantation is the primary mode of treatment for transplant-associated vasculopathy in heart grafts. However, due to the shortage of organs and the decreased short-term survival of patients receiving a second graft, this mode of treatment is not desirable.
Currently, there exists a need for an effective method of treating or preventing chronic graft rejection requiring minimal invasiveness and depletion of the supply of available organs.
Closure of an open wound generally proceeds systematically through the processes of inflammation, repair and closure, remodeling, and final healing (reviewed in Hammar, Int. J. Dermatol., 32(1), 6-15 (1993)). Throughout this sequence, a continuing interaction between diverse cell types is mediated through various intercellular molecules. Notably, cytokines, such as Platelet Derived Growth Factor, Transforming Growth Factor-.beta., and Fibroblast Growth Factor, etc., are important to normal wound closure (i.e., healing) (Id.). Furthermore, arginine metabolism and NO synthesis are increased as a result of wounding (see Shabani et al., Wound Healing Repair and Regeneration, 4(3), 353-62 (1996)).
Many wounds do not complete the healing process. In many patients, such as elderly patients (Kirk et al, Surgery, 114(2), 155-60 (1993), or those suffering from other complications, wounds may persist chronically or wounds may heal incompletely (Hammer, supra, page 6). These patients are more prone to secondary infections or other complications. Several of these other complications, such as in patients undergoing steroid treatment or diabetic patients, are also associated with reduced NO production.
Indeed, supply of exogenous arginine, the catalytic substrate for NOS enzymes, has been demonstrated to accelerate the healing of wounds in animal experiments (Seifer et al., Surgery, 84, 224-30 (1978); Barbul et al., Am. J. Clin. Nutr., 37, 786-94 (1983)). Arginine stimulates wound healing in elderly human patients (Kirk et al., supra), and may act in part through stimulation of NO synthesis (Barbul et al., Surgery, 108(2), 331-37 (1990) (see appended dialog section)). Furthermore, direct topical administration of exogenous NO promotes healing of both chronic and normal wounds (Shabani et al., supra).
Several possible vehicles have been contemplated to deliver NO to wounds to promote healing (discussed in Shabani, supra). Some of these are delivered as pro-drugs, and thus require enzyme activation by means of electron transfer. Furthermore, the solubility of these pro-drugs renders them unlikely candidates for discrete targeting without systemic effects. Other methods involve a synthetic vehicle (a "NONOate") for delivery of NO to the site in question. Many of these are water-soluble and thus are difficult to contain within a wound site. Those which are not water soluble may become progressively less efficient in transferring NO to the wound as the healing process produces new tissue between the NONOate and the wound (Shabani, supra, page 360). Furthermore, use of topical NO delivery to internal wounds would require subsequent surgical invasions to remove the synthetic NO source. Additionally, therapies such as these only deliver one compound to a wound, where natural synthetic pathways leading to NO production also produce other biologically active compounds. Lastly, a constitutive source of exogenous NO may be counter-therapeutic in some applications, as NO is known to cause substantial tissue damage in excessive concentrations.
Thus, there exists a need for a method of promoting the closure or healing of chronic wounds. Additionally, there exists a need for facilitating the closure and healing of internal and external wounds in patients with reduced NO production. Furthermore, there exists a need for facilitating the closure and healing of internal and external wounds in patients with minimal invasiveness and without requiring application of foreign synthetic polymers. Lastly, there exists a need to employ a source of NO and other therapeutically-active compounds to a wound in a manner that prevents oversupply of NO.